Because of its broad potential, gene therapy has been heavily investigated over the past 20 years. However, a clinically viable gene therapy treatment has yet to be realized. The single greatest impediment to the translational development of gene therapy is the lack of safe and efficient means to deliver genetic information to target cells and tissues. While viruses are currently the most efficient way to deliver genetic information, they also pose serious health risks-including immunogenicity and oncogenicity-which have manifested themselves in prior clinical trials. Cationic polymers and lipids have the potential to be non-immunogenic and non-oncogenic delivery vehicles, but many of the available materials are relatively inefficient. Even the most efficient lipids and polymers are orders of magnitude less efficient than viruses, often necessitating the use of micrograms of DNA to achieve transgene expression comparable to that resulting from a virus suspension containing only picograms of genetic material. In order to improve the efficiency of non-viral vectors, they must be designed to overcome extracellular barriers common to all gene delivery vehicles as well as a second set of intracellular barriers encountered once the delivery vehicle reaches the cells of interest. Specifically, 1) the vector must bind to the cell, 2) be internalized, 3) escape from endocytic vesicles into the cytoplasm, 4) move through the cytosol to the nuclear envelope, 5) migrate into the nucleus and 6) release the DNA. Viruses are very efficient because they have evolved specific functions for meeting each of these challenges. Current synthetic materials, however, lack some or all of the functions necessary for efficiently escorting the DNA from outside the extracellular environment into the nucleus. With this R21 application, we propose to develop a reactive cationic ?-helical template for the construction of a library of materials for gene delivery. We are interested in retaining helical architecture throughout the library due to the frequent occurrence of helical domains in many membrane disruptive materials. We believe having this secondary structure as the core feature of the library will yield materials which are able to effectively escape from endocytic vesicles. By subtly changing the hydrophilic/hydrophobic balance of side chains grafted onto the helical backbone, we will generate materials which have a variety of DNA binding strengths. In this manner, we hope to discover polymers with the appropriate balance of DNA binding strength and endosomolytic properties to yield efficient gene delivery. PUBLIC HEALTH RELEVANCE: This R21 application addresses endosomal escape-one of the most important barriers to efficient non-viral gene delivery-through the development of a reactive cationic ?-helical template for the construction of a library of materials for non-viral gene delivery. The cationic helical architecture is believed to aid endosomolysis while subtle changes in the hydrophilic/hydrophobic balance of side chains grafted onto the helical template will allow the identification of polymers with the appropriate balance of DNA binding strength and endosomolytic properties to yield efficient gene delivery.